A New Strategy To Identify Rare Blood Donors: Single Polymerase Chain Reaction Multiplex Snapshot Reaction For Detection Of 16 Blood Group Alleles

Background. As an alternative to phenotyping, large-scale DNA-based assays, which are feasible for high-throughput donor red blood cell typing, were developed for determination of blood group polymorphisms. However, high-throughput genotyping platforms based on these technologies are still expensive and the inclusion of single nucleotide polymorphisms and analysis of the alleles depend on the manufacturer's determination. To overcome this limitation and in order to develop an assay to enable the screening of rare donors, we developed a SNaPshot assay for analysis of nine single nucleotide polymorphisms related to antigens that are difficult to assess using conventional serology. Materials and methods. The single polymerase chain reaction multiplex SNaPshot reaction was optimized to identify nine single nucleotide polymorphisms determining 16 alleles: KEL*3/KEL*4, KEL*6/KEL*7, DI*1/DI*2, DI*3/DI*4, YT*1/YT*2, CO*1/CO*2, DO*1/DO*2, DO*4, DO*5. We designed a single multiplex PCR with primers encompassing the blood group single nucleotide polymorphisms and performed an internal reaction with probe primers able to discriminate the alleles after fragment analysis. The SNaPshot assay was validated with 140 known alleles previously determined by PCR restriction fragment length polymorphism. Results. We were able to simultaneous detect nine single nucleotide polymorphisms defining 16 blood group alleles on an assay based on a multiplex PCR combined with a single base extension using genomic DNA. Discussion. This study demonstrates a robust genotyping strategy for conducting rare donor screening which can be applied in blood centers and could be an important tool for identifying antigen-negative donors and, therefore, for providing rare blood. © SIMTI Servizi Srl.12SUPPL.1s256s263Jungbauer, C., Routine use of DNA testing for red cell antigens in blood centres (2011) Transfus Apher Sci, 45, pp. 61-68Nance, S.T., How to find, recruit and maintain rare blood donors (2009) Curr Opin Hematol, 16, pp. 503-508Veldhuisen, B., Van Der Schoot, C.E., De Haas, M., Blood group genotyping: From patient to high-throughput donor screening (2009) Vox Sang, 97, pp. 198-206Moulds, J.M., Future of molecular testing for red blood cell antigens (2010) Clin Lab Med, 30, pp. 419-429Patnaik, S.K., Helmberg, W., Blumenfeld, O.O., BGMUT: NCBI dbRBC database of allelic variations of genes encoding antigens of blood group systems (2012) Nucleic Acids Res, 40, pp. D1023-D1029Vallone, P.M., Butler, J.M., AutoDimer: A screening tool for primer-dimer and hairpin structures (2004) Biotechniques, 37, pp. 226-231Baleotti Jr., W., Rios, M., Reid, M.E., Dombrock gene analysis in Brazilian people reveals novel alleles (2006) Vox Sang, 91, pp. 81-87Rios, M., Hue-Roye, K., Oyen, R., Insights into the Holleyand Joseph- phenotypes (2002) Transfusion, 42, pp. 52-58Baleotti Jr., W., Rios, M., Reid, M.E., A novel DI*A allele without the Band 3-Memphis mutation in Amazonian Indians (2003) Vox Sang, 84, pp. 326-330Arnoni, C., Latini, F.R.M., Person, R.M., Padronização das técnicas de PCR-RFLP para genotipagem dos alelos KEL*3/ KEL*4 e KEL*5/KEL*6 (2011) Rev Bras Hematol Hemoter, 33 (SUPPL.2), pp. 332-488Baleotti Jr., W., Suzuki, R.B., Ruiz, M., A PCR-RFLP strategy for Wright typing (2011) Rev Bras Hematol Hemoter, 33 (SUPPL. 2), pp. 332-488Brazilian Real - United States Dollar Exchange Rate from Central Bank of Brazil, , http://www4.bcb.gov.br/pec/taxas, April 1st to April 30th, 27/03/2013Daniels, G., The molecular genetics of blood group polymorphism (2009) Hum Genet, 126, pp. 729-742Logdberg, L., Reid, M.E., Zelinski, T., Human blood group genes 2010: Chromosomal locations and cloning strategies revisited (2011) Transfus Med Rev, 25, pp. 36-46Di Cristofaro, J., Silvy, M., Chiaroni, J., Bailly, P., Single PCR multiplex SNaPshot reaction for detection of eleven blood group nucleotide polymorphisms: Optimization, validation, and one year of routine clinical use (2010) J Mol Diagn, 12, pp. 453-460Ferri, G., Pelotti, S., Multiplex ABO genotyping by minisequencing (2009) Methods Mol Biol, 496, pp. 51-58Palacajornsuk, P., Halter, C., Isakova, V., Detection of blood group genes using multiplex SNaPshot method (2009) Transfusion, 49, pp. 740-749Silvy, M., Simon, S., Gouvitsos, J., Weak D and DEL alleles detected by routine SNaPshot genotyping: Identification of four novel RHD alleles (2011) Transfusion, 51, pp. 401-411Silvy, M., Di Cristofaro, J., Beley, S., Identification of RHCE and KEL alleles in large cohorts of Afro-Caribbean and Comorian donors by multiplex SNaPshot and fragment assays: A transfusion support for sickle cell disease patients (2011) Br J Haematol, 154, pp. 260-270Pastinen, T., Kurg, A., Metspalu, A., Minisequencing: A specific tool for DNA analysis and diagnostics on oligonucleotide arrays (1997) Genome Res, 7, pp. 606-614Syvanen, A.C., From gels to chips: "Minisequencing" primer extension for analysis of point mutations and single nucleotide polymorphisms (1999) Hum Mutat, 13, pp. 1-10Information notebook (2011) Blood and Hemoderivates Brasília, , Ministério da Saúde. 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Potencial del aceite de Annona muricata L.: Caracterización nutricional y fitoquímica asociada con no toxicidad

The aim of this study was to evaluate the nutritional quality, phenolic compounds, fatty acid and antioxidant activity in vitro as well as a toxicological screening of A. muricata seed oil in vivo. The chemical composition and quantification of phenolic compounds were determined by the Adolfo Lutz Institute normative. The antioxidant activity was evaluated by DPPH, FRAP and ABTS methods. The oil was extracted by chloroform/ methanol and precipitated crude (AmPtO) and supernatant oils (AmSO) were obtained. The fatty acid profile was evaluated by gas chromatography and total compounds by HPLC-DAD. BALB/C mice received AmPtO and AmSO (0.5 and 1.0mL·Kg-1) for 14 days. Toxicity parameters were assessed. The major fatty acids in the oil were oleic (39.2%) and linoleic (33%). HPLC-DAD suggested the presence of acetogenins (annonacin: 595 [M-H]-), with a greater presence in AmPtO. The AmPtO group showed toxicity, which may be related to the acetogenin content in AmPtO. The AmSO group showed no toxicity and this oil has potential for food or medicinal use.El objetivo de este estudio fue evaluar la calidad nutricional, compuestos fenólicos, ácidos grasos, actividad antioxidante in vitro y evaluación toxicológica del aceite de las semillas de la Annona muricata. La composición química y los compuestos fenólicos se determinaron de acuerdo con las normas del Instituto Adolfo Lutz y la actividad antioxidante por métodos de DPPH, FRAP y ABTS. El aceite fue extraído con cloroformo/metanol obteniéndose un precipitado crudo (AmPtO) y un aceite sobrenadante (AmSO). El perfil de ácidos grasos fue evaluado por cromatografía gaseosa y los compuestos fitoquímicos mediante HPLC-DAD. BALB/C recibieron AmPtO y AmSO (0,5 y 1,0 mL·kg-1) durante 14 días. Se evaluaron los parámetros bioquímicos e histopatológicos. Los ácidos grasos principales fueron oleico (39,2%) y linoleico (33%). HPLC-DAD indicó la presencia de acetogeninas, particularmente anonacina (595 [M-H]-), principalmente en AmPtO. AmPtO presentó toxicidad y esto puede estar relacionado con las acetogeninas. AmSO no presentó toxicidad y tiene potencial para la alimentación o uso medicinal